Vertical Inhibition of the PI3K/Akt/mTOR Pathway for the Treatment of Osteoarthritis

Osteoarthritis is characterized by degenerative alterations of articular cartilage including both the degradation of extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been demonstrated to involve in both processes. Inhibition of its downstream target NF-kB reduces the degradation of extracellular matrix via decreased production of matrix metalloproteinases while inhibition of mTOR increased autophagy to reduce chondrocyte death. However, mTOR feedback inhibits the activity of the PI3K/Akt pathway and inhibition of mTOR could result in increased activity of the PI3K/Akt/NF-kB pathway. We proposed that the use of dual inhibitors of PI3K and mTOR could be a promising approach to more efficiently inhibit the PI3K/Akt pathway than rapamycin or PI3K inhibitor alone and produce better treatment outcome.

Akt, AS160, metabolic risk factors and aerobic fitness in middle-aged women

Aims: This study investigated the association between the basal (rest) insulin-signaling proteins, Akt, and the Akt substrate AS160, metabolic risk factors, inflammatory markers and aerobic fitness, in middle-aged women with varying numbers of metabolic risk factors for type 2 diabetes. Methods: Sixteen women (n = 16) aged 51.3+/-5.1 (mean +/-SD) years provided muscle biopsies and blood samples at rest. In addition, anthropometric characteristics and aerobic power were assessed and the number of metabolic risk factors for each participant was determined (IDF criteria). Results: The mean number of metabolic risk factors was 1.6+/-1.2. Total Akt was negatively correlated with IL-1 beta (r = -0.45, p = 0.046), IL-6 (r = -0.44, p = 0.052) and TNF-alpha (r = -0.51, p = 0.025). Phosphorylated AS160 was positively correlated with HDL (r = 0.58, p = 0.024) and aerobic fitness (r = 0.51, p = 0.047). Furthermore, a multiple regression analysis revealed that both HDL (t = 2.5, p = 0.032) and VO(2peak) (t = 2.4, p = 0.037) were better predictors for phosphorylated AS160 than TNF-alpha or IL-6 (p>0.05). Conclusions: Elevated inflammatory markers and increased metabolic risk factors may inhibit insulin-signaling protein phosphorylation in middle-aged women, thereby increasing insulin resistance under basal conditions. Furthermore, higher HDL and fitness levels are associated with an increased AS160 phosphorylation, which may in turn reduce insulin resistance.

The key regulatory roles of the PI3K/Akt signalling pathway in the functionalities of mesenchymal stem cells and applications in tissue regeneration

Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into various cell types and have been used widely in tissue engineering application. In tissue engineering, a scaffold, MSCs and growth factors are used as essential components and their interactions have been regarded to be important for regeneration of tissues. A critical problem for MSCs in tissue engineering is their low survival ability and functionality. Most MSCs are going to be apoptotic after transplantation. Therefore, increasing MSC survival ability and functionalities is the key for potential applications of MSCs. Several approaches have been studied to increase MSC tissue forming capacity including application of growth factors, overexpression of stem cell regulatory genes and improvement of biomaterials for scaffolds. The effects of these approaches on MSCs have been associated with the activation of the PI3K/Akt signaling pathway. The pathway plays central regulatory roles in MSC survival, proliferation, migration, angiogenesis, cytokine production and differentiation. In this review, we summarize and discuss the literatures related to the roles of the PI3K/Akt pathway in the functionalities of MSCs and the involvement of the pathway in biomaterials-increased MSC functinalities. Biomaterials have been modified in their properties, surface structure and loaded with growth factors to increase MSC functionalities. Several studies demonstrated that the biomaterials-increased MSC functionalities are mediated by the activation of the PI3K/Akt pathway.

Early time course of Akt phosphorylation after endurance and resistance exercise

Purpose The aim of this study was to determine the early time course of exercise-induced signaling after divergent contractile activity associated with resistance and endurance exercise. Methods Sixteen male subjects were randomly assigned to either a cycling (CYC; n = 8, 60 min, 70% V?O2peak) or resistance (REX; n = 8, 8×5 leg extension, 80% one-repetition maximum, 3-min recovery) exercise group. Serial muscle biopsies were obtained from vastus lateralis at rest before, immediately after, and after 15, 30, and 60 min of passive recovery to determine early signaling responses after exercise. Results There were comparable increases from rest in AktThr308/Ser473 and mTORSer2448 phosphorylation during the postexercise time course that peaked 30-60 min after both CYC and REX (P<0.05). There were also similar patterns in p70S6K Thr389 and 4E-BP1Thr37/46 phosphorylation, but a greater magnitude of effect was observed for REX and CYC, respectively (P<0.05). However, AMPKThr172 phosphorylation was only significantly elevated after CYC (P<0.05), and we observed divergent responses for glycogen synthaseSer641 and AS160 phosphorylation that were enhanced after CYC but not REX (P<0.05). Conclusions We show a similar time course for Akt-mTOR-S6K phosphorylation during the initial 60-min recovery period after divergent contractile stimuli. Conversely, enhanced phosphorylation status of proteins that promote glucose transport and glycogen synthesis only occurred after endurance exercise. Our results indicate that endurance and resistance exercise initiate translational signaling, but high-load, low-repetition contractile activity failed to promote phosphorylation of pathways regulating glucose metabolism.

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets × 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNF? protein expression, and IKKSer180/181 and p38MAPK Thr180/Tyr182 phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNF? and IKK Ser180/181. There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). AktSer473 and mTORSer2448 phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k Thr389 increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6Ser235/236 increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.

Exercise-induced phosphorylation of the novel Akt substrates AS160 and filamin A in human skeletal muscle

Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ?7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser 473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr 308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and pp300 were identified as AS160 and filamin A, respectively, with increased phosphorylation (2.0- and 4.9-fold, respectively; P < 0.05) after endurance but not resistance exercise. In conclusion, AS160 and filamin A may provide an important link to mediate endurance exercise-induced bioeffects in skeletal muscle.

Phospho-Akt expression is associated with a favorable outcome in non-small cell lung cancer

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037). These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC. © 2005 American Association for Cancer Research.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

Strategies for co-targeting the PI3K/AKT/mTOR pathway in NSCLC

The PI3K/AKT/mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. We and others have shown that activation of this pathway in non-small cell lung cancer (NSCLC) leads to a more aggressive disease which correlates to poor prognosis for patients. A multitude of selective inhibitors are in development which target key regulators in this pathway, however the success of PI3K targeted inhibition has been hampered by a high rate of innate and acquired resistance. Response to PI3K inhibition may be improved by co-targeting potential mediators of resistance, such as related cell surface receptors or other intracellular signaling pathways which cross-talk with the PI3K pathway. Inhibition of the PI3K pathway may also overcome radioresistance, chemoresistance and immune evasion in NSCLC. The identification of appropriate patient cohorts who will benefit from PI3K co-targeted inhibition strategies will be key to the success of these inhibitors.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Phosphoprotein pathway mapping: Akt/Mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains ∼60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser473 (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr37/46 (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser1108 (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr389 (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.

The effect of lycopene on the PI3K/Akt signalling pathway in prostate cancer

Prostate cancer is common in men with very high mortality which is one of leading causes of cancer-related deaths in men. The main treatment approaches for metastasized prostate cancer are androgen deprivation and chemotherapeutic agents. Although there are initial responses to castration, the resistance to the treatment will eventually occur, leading to castration-resistant prostate cancer. The common chemotherapeutic agents for the treatment of prostate cancer are docetaxel and taxane but outcomes of using these drugs have not been satisfactory. Therefore, it is necessary to find better treatment approaches for prostate cancer and to search for compounds that are effective in prostate cancer prevention. Lycopene extracted from tomato and other fruits or plants such as Gac, watermelon, pink grapefruit, pink guava, red carrot and papaya has been shown to be effective on prostate cancer prevention and treatment. The advantage of the application of lycopene for its anti-prostate cancer activity is that lycopene can reach much higher concentration in prostate tissue than other tissues. In this review, the effect of lycopene on PI3K/Akt pathway is summarised, which could be one of major mechanisms for anti-cancer activity of lycopene.

Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt

Impairment of Akt phosphorylation, a critical survival signal, has been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. However, the mechanism underlying pAkt loss is unclear. In the current study, we demonstrate pAkt loss in ventral midbrain of mice treated with dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), when compared to ventral midbrain of control mice treated with vehicle alone. Thiol residues of the critical cysteines in Akt are oxidized to a greater degree in mice treated with MPTP, which is reflected as a 40% loss of reduced Akt. Association of oxidatively modified Akt with the phosphatase PP2A, which can lead to enhanced dephosphorylation of pAkt, was significantly stronger after MPTP treatment. Maintaining the protein thiol homeostasis by thiol antioxidants prevented loss of reduced Akt, decreased association with PP2A, and maintained pAkt levels. Overexpression of glutaredoxin, a protein disulfide oxidoreductase, in human primary neurons helped sustain reduced state of Akt and abolished MPP+-mediated pAkt loss. We demonstrate for the first time the selective loss of Akt activity, in vivo, due to oxidative modification of Akt and provide mechanistic insight into oxidative stress-induced down-regulation of cell survival pathway in mouse midbrain following exposure to MPTP.-Durgadoss, L., Nidadavolu, P., Khader Valli, R., Saeed, U., Mishra, M., Seth, P., Ravindranath, R. Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt. FASEB J. 26, 1473-1483 (2012). www.fasebj.org

Protumorigenic actions of S100A2 involve regulation of PI3/Akt signaling and functional interaction with Smad3

S100 family of calcium-binding proteins is commonly upregulated in a variety of tumor types and is often associated with tumor progression. Among several S100 members, altered expression of S100A2 is a potential diagnostic and prognostic marker in cancer. Several reports suggest a role for S100A2 in metastasis. Earlier, our studies established regulation of S100A2 by transforming growth factor- (TGF-) and its involvement in TGF--mediated cancer cell invasion and migration. However, the molecular mechanisms of S100A2 protumorigenic actions remain unexplored. In the present study, we demonstrate that overexpression of S100A2 in A549 lung cancer cells induced epithelialmesenchymal transition (EMT) followed by increased invasion, loose colony morphology in soft agar and enhanced Akt phosphorylation (Ser-473). Furthermore, overexpression of S100A2 led to increased tumor growth in immunocompromised mice. In agreement, immunohistochemical examination of resected xenograft tumors established inverse correlation between S100A2 and E-cadherin expression together with activated Akt signaling. Interestingly, our study demonstrates a strong dependence of S100A2 and Smad3 in TGF--induced Hep3B cell EMT and invasion. Most importantly, we demonstrate that these effects of S100A2 are manifested through functional interaction with Smad3, which is enhanced in the presence of high calcium and TGF-. S100A2 stabilizes Smad3 and binds to its C-terminal MH2 domain. Additionally, loss of S100A2 attenuates the transcription of TGF-/Smad3 target genes involved in tumor promotion, such as PA1-1 and vimentin. Collectively, our findings present the first mechanistic details of S100A2 protumorigenic actions and its involvement in TGF--mediated cancer cell invasion and EMT.

Estudio del receptor 5HT2A y de la vía Akt/GSK3 como mecanismos moleculares de la psicosis inducida por el consumo crónico de cannabis

La esquizofrenia es una enfermedad mental de carácter crónico que afecta aproximadamente al 1% de la población , principalmente con edades comprendidas entre los 15 y 4 5 años . Se trata de una enfermedad de inicio en la adolescencia o comienzo de la madurez y cuyo potencial discapacitante persiste y se agrava a lo largo de la vida. La etiología de la esquizofrenia es multifactorial con evidencias de factores genéticos y ambientales. El carácter hereditario de la esquizofr e nia se ha estimado en un 73 - 90% y los factores genéticos que predisponen a la enfermedad son múltiples y heterogéneos. Los estudios epidemiológicos parecen sugerir que el mayor riesgo de desarrollar esquizofrenia es prod ucto de interacciones genético/ ambi entales (predisposición genética a agresiones ambientales) y del fenómeno de epistasis (fenotipo dependiente de la interacción entre diversos genes). Las manifestaciones clínicas de la esquizofrenia se dividen en síntomas positivos, síntomas negativos y dé ficits cognitivos. A causa de los déficits asociados y de su carácter crónico, la esquizofrenia se encuentra entre las diez causas principales de discapacidad por enfermedad del mundo y según la World Health Organization (2001), se estima que es la quinta enfermedad más costosa para la s ociedad en términos de atención requ erida y pérdida de productividad, con un coste anual en la Unión Europea que supera los 35 mil millones de €, según Andlin - Sobocki y Rössler (2005) . A nivel nacional, en el año 2009 los an tipsicóticos atípicos más empleados (risperidona y olanzapina) se encontraron entre los 8 primeros fármacos que más gasto generaron, con un coste económico superior a 350 millones de euros (Sistema Nacional de Salud (2010)) . Además, según el estudio de Pal mer et al. (2005), el riesgo de muerte prematura en la población esquizofrénica es aproximadamente dos veces mayor en la población general, siendo el suicidio la principal causa de este exceso de mortalidad, con una prevalencia estimada entre 5 - 10%. En est e sentido, se ha estimado que entre el 25% y 50% de los pacientes con esquizofrenia cometen por lo menos un intento de suicidio en su vida , según el estudio llevado a cabo por Meltzer (2002) . Dada su prevalencia, la tendencia a la cronicidad y el riesgo su icida de pacientes con esquizofrenia así como los elevados costes socio - sanitarios, la investigación de esta enfermedad es un objetivo prioritario de los sistemas de salud mundiales